I'm using DMEM supplemented with 10% HI FBS and antibiotics for J774A1 cell line. There is no HEPES in DMEM used in our lab. After seeding and serum starvation during 12 h, J774A.1 cells were subjected to hypoxia (12 h) and reoxygenation. I also did not add HEPES to hypoxic serum-free medium.
The LDH levels are too high not only in hypoxic group, but also normoxic group. Because this is my first time to culture J774A.1, I'm not sure what is the problem. J774A.1 are more suceptible to pH and should be cultured in DMEM containing HEPES? or to serum-free condition?
Indeed, it is advised to use HEPES to buffer your medium. The applied percentage of CO2 in the incubator is essential to maintain a specific pH. As mentioned by Alan, if CO2 concentration changes (e.g. if you take out your culture flasks), the equilibrium will shift and pH will rapidly change. Changes in pH will occur less frequent in the presence of HEPES (10 mM can already give good results). If your results with HEPES are identical to your current experiments, then another factor may influence your cells. For example, L-glutamine can be replaced by alanyl or glycyl-glutamine, which is more stable. This will result in lower ammonia concentrations in your medium. Good luck with the experiments!
this cell line is sensitive to PH but this may be due to your serum media that you used. what type of serum free medium did you use?
I've never used this cell line, but you can better understand which is the best medium to use regarding tha ATCC site. Moreover, if in your medium Hepes is absent, i usually add hepes to the medium to mantain the correct pH, if you don't have Hepes is very difficult to mantain the correct pH in your cell culture!
I think that this the main problem you have!
If you grow cells under anaerobic conditions, then glucose is converted to lactic acid rather than carbon dioxide and water. HEPES buffer should avoid this, or you should check the pH of your medium. In the leishmaniasis field we tend to use RPMI1640 rather than DMEM for macrophage culture.
As the recommended DMEM contains 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate, I used same DMEM to make serum-free medium. It sounds very foolish and the beginner's idea, I would like to ask a question. Should the pH of medium be always maintained in all in vitro experiments? As the goal of hypoxic or anaerobic culture is to verify same situation in vivo, I keep being troubled with using HEPES and serum for hypoxic medium. As Dr. Fairlamb said, the acidosis occurs during ischemia and I'm not sure it can be prevented using supplement such as HEPES. Moreover, there were rare references mentioning about using HEPES in hypoxic experiment. Is it possible they used HEPES without mentioning in their works?
I am always very grateful for your advice.
Sincerely,
Hyo-Yeon Kim
Sodium bicarbonate is a buffering agent, provided there is an atmosphere of 3-5% carbon dioxide. If not, bicarbonate will pick up hydrogen ions forming carbonic acid which dissociates releasing carbon dioxide into the gas mixture. The net result is that the culture medium becomes more alkaline. This is also observed when filtering media under negative pressure. Having 20 mM HEPES, or another Good buffer such as TES, present ameliorates this effect. Sigma sells many versions of DMEM, some of which contain HEPES, others that do not, so it is possible that authors have not been specific in describing their method.
Indeed, it is advised to use HEPES to buffer your medium. The applied percentage of CO2 in the incubator is essential to maintain a specific pH. As mentioned by Alan, if CO2 concentration changes (e.g. if you take out your culture flasks), the equilibrium will shift and pH will rapidly change. Changes in pH will occur less frequent in the presence of HEPES (10 mM can already give good results). If your results with HEPES are identical to your current experiments, then another factor may influence your cells. For example, L-glutamine can be replaced by alanyl or glycyl-glutamine, which is more stable. This will result in lower ammonia concentrations in your medium. Good luck with the experiments!
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